sign in sign up
<<
Home , Proanthocyanidin

Arabian Journal of Chemistry (2020) 13, 1105–1119

King Saud University Arabian Journal of Chemistry

www.ksu.edu.sa www.sciencedirect.com

ORIGINAL ARTICLE Composition and biological activity of the Algerian plant Rosa canina L. by HPLC-UV-MS

Samira Fetni a,b,c, Nabil Bertella a,*, Ammar Ouahab d,*, Jose Miguel Martinez Zapater b, Sonia De Pascual-Teresa Fernandez c a Department of Biology of Organisms, Faculty of Sciences of Nature and Life, University of Batna 2, Algeria b Institute of Science of Vine and Wine (CSIC), Spain c Department of Metabolism and Nutrition Institute of Food Science and Technology and Nutrition (Ictan-csic), Spain d Department of Pharmacy, Faculty of Medical Sciences, University of Batna 2, Algeria

Received 12 July 2017; accepted 25 September 2017 Available online 6 October 2017

KEYWORDS Abstract The present study was carried out in order to identify and characterize the compounds of Ethanolic extract; Rosa canina fruits by HPLC-UV-MS. The total phenolic determiner by a new Fast Blue method HPLC-UV-MS; (FBBB), which detects phenolic directly, reported an average total phenolic concentration of 1.7 Antioxidant activity; folds greater than Folin-Ciocalteu (F-C), which indicates that an indirect detection method of total Oxidant effect; phenolic should be replaced in future studies by the FBBB method. TPC of the ethanolic extract Bilobalide A was positively correlated with 1,1-diphenyl-2-picryl-hydrazyl (DPPH) free radical scavenging effect.

The DPPH activity of R. canina extract which is higher than the IC50 of the ascorbic acid and Buty- lated Hydroxytoluene (BHT), but lower than the IC50 of and trolox. The determination of intracellular reactive oxygen species (ROS) proved the antioxidant effect of the extract on HepG2 and SH-SY5Y cells. A concentration of 1.63 lg/ml on HepG2 cells had an oxidizing effect instead of the antioxidant effect, which is due to the existence of a tert-butyl group in sesquiterpene iden- tified by HPLC-UV-MS method. These results indicate that the fruits of R. canina can be used as a natural source of antioxidants against oxidative stress and some types of cancer. Ó 2017 The Authors. Production and hosting by Elsevier B.V. on behalf of King Saud University. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

1. Introduction

R. canina belongs to the family of Rosaceae and genus Rosa, * Corresponding authors. with about 200 species spread in the temperate zone and sub- E-mail addresses: [email protected] (N. Bertella), ouahab. tropics of the Northern hemisphere, R. canina pseudo-fruit was [email protected] (A. Ouahab). traditionally used in preventive therapy and for the prepara- Peer review under responsibility of King Saud University. tion of some foods such as jam, beverages and probiotic drinks (Montazeri et al., 2011). Natural substances and plants in general, are an immense Production and hosting by Elsevier source of chemiodiversity, often with very original structures https://doi.org/10.1016/j.arabjc.2017.09.013 1878-5352 Ó 2017 The Authors. Production and hosting by Elsevier B.V. on behalf of King Saud University. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). 1106 S. Fetni et al.

Fig. 1 Calibration curve of total phenolic content (TPC) of ethanolic extract of Rosa canina fruits using fast blue method (1A), and Folin-Ciocalteu (1B).

The aim of the present study is to identify and characterize Table 1 Total phenols contents in ethanolic extract R. canina the main antioxidants in the ethanolic extract of R. canina fruits. fruits using HPLC–UV–MS. Additionally, the antioxidant Content (lg/mg DM) FBBB F-C Extraction and cytotoxic effects of R. canina extract are also explored. Total phenols yield (%) 30% ethanol 598.25 354.46 20 2. Materials and methods ± 0.49 ± 0.05 Data expressed in lg equivalent of gallic acid (GAE) to 1 mg of 2.1. Samples methanolic extract of Rosa fruits. The fruits of R. canina were harvested from the region of Batna, Algeria, in October 2016. The samples were washed, that are usually impossible to synthesize (structural complex- then dried at room temperature and ground before storing at ... ity, stereospecificity ). , and terpenoids 20 °C. are the most essential bioactive identified in R. canina fruits by HPLC–UV–MS method. They are a wide 2.2. Standards and reagents range of secondary compounds distributed in various plants and play an important role in normal growth and defence. All chemicals and reagents used in this study were of analytical They display various structures and a broad range of biologi- grade and were obtained from Sigma-Aldrich chemistry cal activities (Naczk and Shahidi, 2004). (Madrid, Spain). HPLC grade solvents were purchased from Most qualitative and quantitative analyses of phenolics are Merck Darmstadt, Germany). The HPLC grade water was usually traditional methods such as HPLC-UV for qualitative prepared using a Milli-Q system (Millipore Lab., Bedford, analysis and Folin-Ciocalteu method (F-C), which is the only MA, USA). procedure used to measure total phenolics through the reduc- tion capacity of the components of the extract. Whereas, the 2.3. Preparation of the phenolic extracts novel total phenolic method utilizing Fast Blue BB diazonium salt is based on the coupling of phenolic compounds with the diazonium. The coupling mostly occurs para to the phenolic The extraction of the polyphenols is carried out three times activating group, unless the position is already occupied. For by mixing 50 ml of ethanol/water (70:30; v/v)/plant powder R. canina extract, the Fast Blue (FBBB) method had higher 50 mg. The extract was left in the ultrasonic bath (FALC gallic acid equivalents (GAE) values than the standard Instruments, Italy) for 30 min after centrifugation at Folin–Ciocalteu in the dosage of total phenolic (Maria et al., 5000 rpm for 20 min at 10 °C and the ethanol was removed 2017; Medina, 2011a,b). in vacuum using a rotary evaporator Temperature below There are many analytical methods available to assess the 40 °C. In order to remove the apolar molecules contained antioxidant capacity such as DPPH scavenger effect, which is in the extract, a second extraction was performed by using the method used to evaluate the antioxidant activity in vitro. 4 ml of the hexane/water mixture at a ratio of 50:50; v/v, Cellular antioxidant activity by determination of intracellular and then mixed with the resulting ethanolic extract of the reactive oxygen species (ROS) is an approach used to evaluate first extraction. The operation was repeated three times, the antioxidant activity based on cells (Yin et al., 2019). The and then the extract was dried under vacuum by heat phenolic compounds present in R. canina made it one of the applied in the Savant Speed VacThermo Scientific preferred plants in phyto-therapy and in the pharmaceutical Concentrator SPD131DDA for 4 h. The resulting extract sector. R. canina presents a cytotoxic effect following an was lyophilized in a laboratory lyophilizer (BETA 2-8 LD MTT test. This effect prevented from cancer by several mecha- plus - Martin Christ) for 24 h. nisms. They decrease both cell proliferation and oxidative Extraction yield (%) = weight of extract obtained 100/ stress, block cell cycles, and induce apoptosis (Ren et al., 2003). weight taken of the material vegetal (Baghdikian et al., 2016). Composition and biological activity of the Algerian plant 1107

Fig. 2 HPLC-UV-DAD chromatograms of extract of Rosa canina fruits.

2.4. Determination of total phenols contents by Folin-Ciocalteu at a concentration of 0.5 mg/ml. Briefly, 10 ll of the sample (F-C) and Fast Blue BB (FBBB) methods was transferred into a well in a 96-well plate and 150 lLof 6% Folin–Ciocalteu reagent was added and mixed, after 3 l The total phenolic content was evaluated by F-C method min, add 50 l/well of saturated sodium bicarbonate solution (Khanam et al., 2012). The ethanolic extracts were prepared (0.6 M) was added and mixed gently. Incubate the plate for 1108 S. Fetni et al.

2 h at room temperature and in the dark and read the absor- 2.5. Preparation of sample the HPLC-UV-MS analysis bance at 725 nm by the BioTek Synergy HT multi-mode microplate reader (BioTek Instruments Inc., Vermont, USA) 50 mg of the dried paste was mixed with 10 ml of water/ and the data was acquired and processed using BioTek’s -0.5% HCl (80:20; v/v). The mixture was placed in Gen5TM software (BioTek Instruments Inc.). the ultrasonic bath for 30 min (FALC Instruments, Italy), then The total phenolic content was determined by FBBB the extract was centrifuged at 5000 rpm for 20 min at 10 °C. method (Lester et al., 2012). The ethanolic extracts were pre- The operation was repeated three times and the three succes- pared at a concentration of 0.25 mg/ml. The reaction started sive acidified methanolic supernatants were evaporated to dry- m by adding 150 l/well of sample in the 96-well plate, and then ness under vacuum without heat applied to the SPV131DDA l l mixed with 15 l/well of Fast Blue (0.01% v/v H2O) and 15 l/ Thermo Scientific SPD131DDA Concentrator for 3 h. The well of NaOH (5% v/v H2O). After incubating the plate for samples were reconstituted with 200 ll of formic acid 0.1%, 120 min at room temperature, the absorbance was read at where the sample was centrifuged for 10 min at 9000 rpm, 420 nm with a BioTek Synergy HT multi-mode microplate and then the supernatant was filtered and diluted (1:10) and reader (BioTek Instruments Inc., Vermont, USA), and data finally stored in a flask at 20 °C until analysis (Mraihi was acquired and processed using BioTek’s Gen5TM software et al., 2015). (BioTek Instruments Inc.). A calibration curve was established for the two methods using a standard solution of gallic acid (0.6–0.025 mg/ml) and 2.6. Preparation of standard solution for HPLC-UV analysis expressed as lg of gallic acid equivalents (GAE)/mg of dry weight (dw). For F-C method, y = 1.2943x + 0.0877; R = 0.997, and For the preparation of the standard calibration curves, the for FBBB method, y = 1.1869 + 0.0267; R = 0.999. stock solutions of , gallic acid, protocatechuic Where x is the total phenolic content of ethanolic extract. A acid, chlorogenic acid, caffeic acid, p-coumaric acid, ferulic control was carried out with water, which followed the same acid, , epicatechin, quercetin, quercetin-3-glucoside, treatment as the extract. , resveratrol and were prepared informic

Fig. 3 HPLC-UV-DAD calibration curves of phenolic standards, Apigenin (3A), Cafeic acid (3B), Catechin (3C), Chlorogenic acid (3D), Ferulic acid (3E), Epicatechin (3F), Gallic acid (3G), Kaempferol (3H), p-coumaric acid (3I), Phloroglucinol (3J), Protocatechuic acid (3K), Quercetin (3L), Quercetin-3-glucoside (3M), Resveratrol (3N). Composition and biological activity of the Algerian plant 1109

Fig. 3 (continued) acid 0.1% at a concentration of 1 mg/ml. The concentrated 2.8. HPLC–MS analysis solutions were then diluted with 0.1% formic acid to obtain l 1, 3, 6, 12, 25, 50 and 100 g/ml. All solutions were filtered In order to identify and characterize the phenolic com- l through a 0.2 m sartolon polyamide membrane filter. pounds, a sample of 10 lL of extract was analyzed using HPLC with detection Agilent 1200 Series 2.7. Analysis of polyphenols by HPLC-UV liquid chromatography equipped with an electrospray atmo- spheric pressure (ESI) and employing an ESI (electrospray 20 ll of the sample was placed in an Agilent 1200 Series HPLC ionization), phenomenex Luna C18 (150 4.6 m 3 lm) was for analysis. The Ultrabase column (C18.5 lm, 150 4.6 mm) used with a flow rate of 0.5 ml/min. The capillary tension was used at a flow rate of 0.5 ml/min coupled to a diode array was 3 V, and the capillary temperature was 180 °C. detector (DAD) and a quaternary pump. The temperature of Mobile Phase A was 0.1% Formic Acid in water, while the column was set at 25 °C and the stopping time was 55 Mobile Phase B was 0.1% Formic Acid in Acetonitrile min. The solvents used for the analysis were formic acid (% B: 0 min = 10%, 30 min = 30%, 35 min = 35%, 0.1% (A), acetonitrile with formic acid 0.1% (B), ultrapure 40 min = 45%, 50 min = 10%). The spectra were recorded water (C) and acetonitrile (D) (Pallaufa et al., 2008). in negative ion mode and the MS detector was programmed 1110 S. Fetni et al.

Table 2 Identification of certain phenolic compounds in the extract of Rosa canina L. fruits by LC-UV. Compounds Concentration (lg/ml) kmax Rt (min) Area Equation Coefficient R Phloroglucinol 100 280 5.887 505.37 y = 0.0175x 1.2643 0.992 Gallic acid 50 280 4.886 2239.65 y = 0.0176x 8.1344 0.992 Protocatechuic acid 50 280 10.658 546.69 y = 0.0486x + 0.1167 1.000 Chlorogenic acid 50 280 12.392 1117.06 y = 0.0277x 0.3489 0.999 Caffeic acid 50 280 15.404 1399.72 y = 0.0268x 1.1533 0.999 p-coumaric acid 50 280 21.762 649.97 y = 0.0248x 0.2303 1.000 Ferulic acid 50 280 20.809 1002.28 y = 0.0185x 3.4214 0.997 Catechin 100 280 11.392 518.73 y = 0.0533 + 2.3051 0.997 Epicatechin 100 280 13.666 716.76 y = 0.0659x 4.7479 0.976 Quercetin 100 280 33.37 57.78 y = 0.0243 + 6.862 0.990 Quercetin-3-glucoside 100 280 19.491 508.84 y = 0.0326x 0.7688 1.000 Apigenin 100 330 38.642 25.37 y = 0.0029 + 8.2269 0.974 Resveratrol 15 360 31.299 305.4 y = 0.0661x 0.0455 0.998 Kaempferol 100 360 44.44 44.237 y = 0.0326 + 2.9976 0.988

Fig. 4 HPLC-MS-DAD chromatograms of extract of Rosa canina fruits at-ESI mode. to perform a consecutive scan series: extended dynamic 2.10. Cell culture and extract treatments range, low 1700 m/z (Pallaufa et al., 2008). HepG2 human hepatoma cells, SH-Sy5y human neuroblas- 2.9. DPPH radical-scavenging activity toma were seeded and cultured regularly in DMEM medium and 10% fetal bovine serum, but they were changed to Antioxidant activity was determined by DPPH free radical serum-free medium 24 h prior to testing. Cells were treated scavenging effect by using quercetin, ascorbic acid, trolox with different concentrations of ethanolic extract (0.01–250 l and BHT as positive control, Crude extract was prepared with g/ml) within 24 h. a range of concentrations (10–600 lg/ml). 290 lL of methano- lic DPPH solution (1 105 M) was added and the mixture was 2.11. Determination of ROS incubated for 30 min at room temperature. The absorbance was read at 517 nm and the data were acquired and Cellular oxidative stress was quantified by the dichloroflu- processed using BioTek’s Gen5TM software (BioTek orescence test (DCFH). DCFH becomes dichlorofluores- Instruments Inc.). Free radical scavenging activity was cein (DCF) after being oxidized by intracellular determined according to the equation % Antioxidant Activity oxidants, and emits fluorescence. By quantifying the (AA) = 100-[{(Abssample-Absblank) 100}/Abscontrol] published fluorescence at an excitation wavelength of 485 nm and previously (Nwaehujor et al., 2014). an emission wavelength of 530 nm, a fair estimate of Composition and biological activity of the Algerian plant 1111 the overall oxygen species generated under the various 2.12. Statistical analysis conditions was obtained by using fluorescent probe ° (DCFHDA) after incubation in CO2 at 37 C for 30 The data were expressed as mean ± DS. Significant min (Granado-Serrano et al., 2006). differences were calculated by testing linear trends using

Fig. 5 MS/MS spectra of chemical compounds of extract of Rosa canina. Apigenin (5A), Cafeic acid (3B), Apigenin-7-O-glucoside (5B), Bilobalide A (5C), Catechin (5D), Citric acid (5E), Dihydroquercetin (5F), Ellagic acid (5G), Gallacetophenone (5H), (5I), Kaempferol-7-O-glucoside (5J), Luteolin 5-methyl ether (5K), Luteolin-40-O-glucoside (5L), Luteolin-7-O-glucoside (5M), B3 (5N), Procyanidin B6 (5O), Pyrogallol-2-O-glucuronide (5P), Quercetin dehydrate (5Q), Quercetin (5R), Quercetol 3-O-rutinoside (5S), Rosmarinic acid (5T). 1112 S. Fetni et al.

Fig. 5 (continued) single-factor analysis (ANOVA), using Dunnett’s multiple 3. Results and discussion comparison test. The data used for the IC50 determina- tion were examined using a nonlinear regression sigmoid 3.1. Total phenolic contents (TPC) in R. canina fruits fit using the GraphPad Prism. Differences with P < .05 were considered significant. The SPSS version 20.0 Our results indicate that the FBBB test provides a higher and program was used. more accurate estimate of total phenolic due to its direct Composition and biological activity of the Algerian plant 1113

Fig. 5 (continued) reaction with phenolic compounds in R. canina fruits as well as fruit than in the total phenolics analyzed by FBBB assay (598. the FBBB reaction with gallic acid substrate was highly linear 25 ± 0.49 lg GAE/mg DM). Results are presented in Table 1. (R = 0.999). Accordingly, the F-C assay had a positive linear The FBBB test, which detects phenolics directly, reported an response to gallic acid (R = 0.997) Fig. 1. Although the total average 1.7 fold higher concentration of total phenolics than phenol concentration of F-C expressed as the value (354.46 F-C. Previous studies of fruit using the F-C assay ± 0.05 lg GAE/mg DM), these values were lower in the same have greatly underestimated the total phenolic concentration, 1114 S. Fetni et al.

Fig. 5 (continued) and this assay should be replaced in future studies by the Fast absorption spectrum of reference standards (phloroglucinol, Blue BB assay (Medina, 2011a,b). gallic acid, protocatechuic acid, chlorogenic acid, caffeic acid, p-coumaric acid, ferulic acid, catechin, epicatechin, quercetin, 3.2. HPLC-UV analysis quercetin-3-glucoside, apigenin, resveratrol and kaempferol). From the HPLC-UV profile it was observed that the ethanolic Qualitative analysis of the ethanolic extract of R. canina fruits extract showed the presence of four big peaks at 280 nm. The was tested by using HPLC-UV analysis and their chromato- peaks were identified as gallic acid, chlorogenic acid, caffeic graphic profile was compared with the retention times and acid and ferulic acid with Rt between 4.886 and 20.809 min. Composition and biological activity of the Algerian plant 1115

Table 3 Identification of certain phenolic compounds in the extract of Rosa canina L. fruits by LC-MS. No Proposed compound Rt Accurate mass MS/MS (m/z) Peak purity Molecular Nature of compound (min) [M-H] (%) formula

01 Citric acid 6.41 191.022 191 79.71 C6H8O7 Tricarboxylic acid 02 Gallacetophenone 6.56 167.0358 125, 167 77.41 C8H8O4 Hydrolysable 03 9.13 577.1375 425, 407, 289. 91.66 C30H26O12 Proanthocyanidin 287

04 2,3-Digalloylglucose 9.26 483.0815 483, 125 55.19 C20H20O14 glucoside

05 Pyrogallol-2-O-glucuronide 10.06 301.0582 301, 125 81.95 C12H14O9 Hydrolysable tannin glucoside

06 11.39 577.1375 425, 407, 289, 91.66 C30H26O12 Proanthocyanidin 287

07 Catechin 12.76 289.0754 289 68.36 C15H14O6 -3-ol 08 Gallocatechol 13.02 305.0664 305, 289 71.21 C15H14O7 Flavan-3-ol 09 Quercetin 3,7-diglucoside 18.43 625.1462 625, 463, 301 47.89 C27H30O17 Flavonol glucoside 10 Procyanidin B6 18.93 577.1382 425, 407, 289, 87.09 C30H26O12 Proanthocyanidin 287

11 Quercetin dihydrate 18.97 337.0601 337, 301 nd C15H14O9 Flavonol 12 Bilobalide A 20.48 325.0914 325 91.1 C15H18O8 Sesquiterpene 13 Luteolin 5-methyl ether 20.50 299.0542 299, 285 67.16 C16H12O6 Flavone 14 Quercetol 3-O-rutinoside 22.47 609.1483 609, 463, 301 93.23 C27H30O16 Flavonol glucoside 15 Ellagic acid 23.46 301.0002 301 95.34 C14H6O8 Hydrolysable tannin 16 Kaempferol-7-O-glucoside 24.11 447.0942 447, 285 67.18 C21H20O11 Flavonol glucoside 17 Dihydroquercetin 26.16 303.0520 303 96.65 C15H12O7 Flavonol 0 18 Luteolin-4 -O-glucoside 27.63 447.0944 447, 285 96.92 C21H20O11 Flavone glucoside 19 Apigenin-7-O-glucoside 28.40 431.1004 431, 269 68.94 C21H20O10 Flavone glucoside 20 Rosmarinic acid 30.94 359.0755 359 82.78 C18H16O8 Hydroxycinnamic acid 21 Luteolin-7-O-glucoside 32.19 447.0939 447, 285 85.61 C21H20O11 Flavone glucoside 22 Quercetin 36.48 301.0358 301 95.14 C15H10O7 Flavonol 00 23 Kaempferol-3-Glucoside-2 -p- 38.31 593.1311 593, 463, 285, 98.27 C30H26O13 Flavonol glucoside coumaroyl 163

24 Kaempferol 39.13 285.0411 285 97.12 C15H10O6 Flavonol 25 Apigenin 43.88 269.0462 269 83.54 C15H10O5 Flavone nd: not detected.

Fig. 6 Reduction indexes of the DPPH scavenging effect (%) versus concentration, each value in the graph is shown as means ± SD. 1116 S. Fetni et al.

Table 4 IC50 values of the DPPH free radical scavenging of Rosa canina fruits. Samples Rosa extract Ascorbic acid Quercetin BHT Trolox

DPPH scavenging activity (IC50 lg/ml) 156.74 ± 0.56 46.74 ± 1.14 157.45 ± 2.11 132.22 ± 0.64 163.55 ± 1.25 Positive controls and Rosa extract were done in triplicate (n = 3).

Fig. 7 Effect of ethanolic extract on intracellular ROS levels in HepG2 (7A), and SH-SY5Y cells (7B). *P < .05, ***P < .001 significantly different from negative control cells and positive control cells.

Phloroglucinol, protocatechuic acid, p-coumaric acid, Cate- HPLC-UV-MS/MS analysis using negative ionization modes, chin, epicatechin, quercetin-3-glucoside and resveratrol were results are shown in Fig. 4, the identities, retention times identified at 280 nm with Rt between 5.887 and 31.299 min, (Rt min), chemical formula, pseudomolecular ions [M-H], and traces of querce´ tine, apigenin and kaempferol Figs. 2 peak purity (%) and fragment ions MS/MS (m/z) for individ- and 3 (Table 2). This analysis gives an idea on the composition ual compounds were reported in Fig. 5 (Table 3). of polyphenols presented in the fruits of R. canina (Jafri et al., The phenolic acids with its types (Hydroxycinnamic acid 2017). and Tricarboxylic acid) were represented by peaks 1 and 18 in the chromatogram, and they were identified as citric acid 3.3. HPLC-MS analysis of phenolic compounds and peaks and rosmarinic acid, respectively. identification Citric acid was identified at Rt 6.41 min and m/z 191.022, the important role of this acid in the Krebs cycle has drawn 25 phenolic compounds belonging to a variety of classes the attention of a number of research groups, especially for of natural products were detected in R. canina by use in diagnosis and targeting of cancer. Rosmarinic acid, Composition and biological activity of the Algerian plant 1117 which is the print of Rosaceae family, corresponded to Rt fragmentation of O-glycosylated flavonols. The releasing of 30.94 min and m/z 359.0755 as shown in (Table 3)(Mcdunn monosaccharide residue is shown by the loss of sugar et al., 2013; Tohma et al., 2016). (Mraihi et al., 2015). Flavan-3-ol peaks were represented by peaks 6 and 7at Rt The MS/MS fragmentation mechanism of flavonolsagly- 12.76, 13.02, and m/z 289.0754, 305.0664 respectively, which cone based on cleavage of two CAC bonds of the C-ring corresponded to catechin and gallogatechol. Proanthocyanidin releasing two fragment ions which give informations about B-type isomers were detected in three peaks (2, 5 and 9) at the number and type of substituents (Cuyckens and Claeys, 9.13, 11.39 and 18.93 min and the signal at m/z 577 indicated 2004), the pseudomolecular ions [M-H] of flavonolsaglycone that the dimers are mixed; two units of catechin and/or epicat- identified in R. canina at m/z 625, 609, 593, 447 and 463. Fur- echin. The obtained MS/MS (m/z) fragments of the condensed thermore, Therefore the product ions, [aglycon–H]-, were tannins were 425, 407, 289 and 287 (Friedrich and Eberhardt, reported at m/z 285 (Table 3), were shown fragmentations of 2000). The fragmentation pathway by Retro Diels-Alder in the flavonols peaks 16 and 23 (same pseudomolecular ion [M- T-unit of procyanidin B3 was the important mechanism that H] at m/z 447 and 593, respectively) corresponding to allowed its detection in the ions m/z 425 and 407. Hydrolysable kaempferol-7-O-glucoside, kaempferol-3-Glucoside-200-p-cou tannins (gallotannin and ellagitannin; gallacetophenone and maroyl. The releasing fragments of the peaks 9 and 14 corre- ellagic acid respectively) were reported by two peaks (2 and sponded to the loss of dihexosyl (glucose and/or galactose 15). Hydrolysable tannin glucosides were assigned to peaks 3 = 162 lma) and rhamnosyl hexosyl (146 to 162 lma) to and4, which corresponded to 2,3-Digalloylglucose and obtain the quercetin aglycon, dihydroquercetin and quercetin Pyrogallol-2-O-glucuronide; they were identified at 6.56, dehydrate at m/z 301.303 and 337 respectively. These disaccha- 9.26, 10.06 and 23.46 min with m/z 167.0358, 483.0815, ride should be either a diglucoside = Glc-Glc (m/z 324) or a 301.0582 and 301.0002 respectively (Table 3)(Kassim et al., Gal-Gal (m/z 324) and a rutinoside = Glc-Rha (m/z 308) or 2010; Ridder et al., 2012). a Gal-Rha (m/z 308) respectively linked to the quercetin agly- The chromatogram peak 11 reflected the different types of cone by either Glc or Gal sugar. So these peaks corresponded terpenoids such asbilobalide A. These compounds were identi- to quercetin 3,7-diglucoside and quercetol 3-O-rutinoside at m/ fied at Rt 20.48 min and m/z 325.0914 with high peak purity z 625 and 609, respectively. 91.1% (Table 3)(Rana et al., 2014; Wang et al., 2013). The principal subclasses of flavonoids were the abundant compounds present in R. canina (Table 3), in the structure por- 3.4. DPPH radical-scavenging activity trayal, we initially judged if the flavonoid is a C- glycosylated. The carbon-carbon obligation of C-glycosyl fla- The antioxidant activity of the phenolic compounds depends vonoids is impervious to burst and in C- for the most on their chemical composition and structural conformation. part the fracture of the sugar unit is watched. Fracture path- These antioxidants react with DPPH, diminishing a number way of O-glycosylated flavonoids begins with the cleavage of of DPPH molecules equivalent to the quantity of accessible the glycosidic bonds and disposal of the sugar moieties with hydroxyl groups (Chaouche et al., 2014; Shimada et al., charge maintenance on aglycone. In mixes containing at least 1992). The ethanolic extract and the standards (trolox, querce- two sugars to the same aglycone carbon, particles emerging tin, BHT and ascorbic acid) were tested and expressed in per- from the cleavage of the glycosidic bonds between sugar units centage of reduction of DPPH radicals in a dose-dependent are feeble. Despite the fact that the aglycone and the glycane manner. For instance, At 600 and 400 lg/ml the ethanolic were altogether recognized, the exact structure of the flavo- extract resulted in 77.22 and 68.33% reduction, respectively. noids glycoside couldn’t be constantly decided on the grounds Meanwhile, ascorbic acid at concentrations ranging between that personality and the site of association of monosaccharide 600 and 400 lg/ml resulted in 91.61 and 91.49% reduction. can’t be controlled by LC-MS. The structures of mixes were at Quercetin on the other hand displayed 88.78 and 85.39% last distinguished by correlation with bibliographical studies reduction. BHT resulted in 88.44 and 88.19% reduction. While (Ana Plazonic´ et al., 2009). trolox displayed 90.99 and 89.80% reduction. Results are The C-glycosylated flavones were additionally found in R. shown in Fig. 6. This extract had a high activity compared canina fruits. Peaks 18 and 21 corresponded to two isomers, with each of the standards because it was rich in phenolic com- namely, luteolin-40-O-glucoside and luteolin-7-O-glucoside pounds such as methyl 4-O-galloylchlorogenate that showed (Table 3). Their identities were based on their pseudomolecular high antioxidant activity in the DPPH free-radical assay. The ion [M-H] at m/z 447 and MS/MS spectra. They gave out high antioxidant capacity of this compound suggested that this fragments assigned to the losses of a hexosyl (glucose = 16 plant was a good protection against oxidative damage (Ma 2 lma) to obtain the luteolin aglycone at m/z 285 and luteolin et al., 2003). The genkwanin and hydroxygenkwanin exhibited 5-methyl ether at m/z 299, and from an apigenin-7-O-glucoside significant anti-oxidative effect on DPPH (Ren et al., 2013). at m/z 431 giving an apigenin aglycon at m/z 269 (Fernandes The DPPH radical scavenging effect was measured for some et al., 2017). terpenoids and derivates of glycerol (Talla et al., 2016). The are the principal compounds present in R. can- antioxidant activities displayed as IC50 values are illustrated ina fruits; they differed in the number and position of pheno- in (Table 4). The IC50 value of ethanolic extract was higher lic hydroxyl AOH. Flavonols were frequently found in the than that of the ascorbic acid and BHT, but lower than that form of heteroside in plants are potent antioxidants that of quercetin and trolox. This difference was due to the richness serve to protect the plant from reactive oxygen species of the extract in flavones (isoorientin and wogonin), glycosy- (ROS) (Nakabayashi et al., 2014), MS/MS spectra discharg- lated derivatives of isoflavone glucoside ( and cavi- ing fragments relating to the misfortunes of sucre, the glyco- unin), and tannins that were detected by HPLC-MS. The sidic bond was cleaved about the mechanism of important properties of the abundant antioxidants in R. canina 1118 S. Fetni et al. fruits conferred dismutation of free radical DPPH. The mech- were due to the presence of flavonoids, tannins, terpenoids, anism by which some hydroxyl groups were connected with and glycerol glucoside. The of hydrogen atoms of phenolic compounds to become stable dia- R. canina fruits made the plant a rich source of antioxidants. magnetic molecules (Boutennoun et al., 2017; Yang et al., The high antioxidant capacity of R. canina extract suggested 2016). that this plant could be used as an additive in the food indus- In the present study, the positive correlation between the try; providing good protection against oxidative damage. It quantity of antioxidants in R. canina and the DPPH free rad- can also be used as an effective natural treatment to control ical activity (R = 0.9962) was proved (Aksoy et al., 2013; several types of cancer. The beneficial effects of R. canina fruits Tchouya and Barhe, 2016). can lead to its usage in diets with nutritional and healthy advantages. 3.5. Determination of intracellular ROS levels

References HepG2 and SH-SY5Y cells are the very useful model for studying the effect of oxidative stress, it is demonstrated that Aksoy, L., Kolay, E., Agˆ ilo¨ nu¨ , Y., Aslan, Z., Kargiogˆ lu, M., 2013. the cytotoxic effect of tert-butyl-induced exogenous oxidative Free radical scavenging activity, total phenolic content, total stress (t-BOOH) trigger deleterious effects on cell growth antioxidant status, and total oxidant status of endemic Thermopsis resulting both from a rapid inflow of the free radical generator turcica. Saudi J. Biol. Sci. 20, 235–239. into the cell. As a result the induction of transient increases in Al-Gubory, K.H., Fowler, P.A., Garrel, C., 2010. The roles of cellular cytosolic Ca2+ to regulate the cellular process by transcription reactive oxygen species, oxidative stress and antioxidants in factors that regulates the expression of genes in response to pregnancy outcomes. Int. J. Biochem. Cell Biol. 42, 1634–1650. oxidative stress and kept cell survival. The correlation is Baghdikian, B., Filly, A., Fabiano-Tixier, A.S., Petitcolas, E., reversed between the high concentration of tert-butyl with Mabrouki, F., Chemat, F., Ollivier, velyne, 2016. Extraction by the amount of cytosolic Ca2+ obtained during the shock oxi- solvent using microwave and ultrasound-assisted techniques fol- dation and when the excessive or unregulated levels of Ca2+ lowed by HPLC analysis of Harpagoside from Harpagophytum procumbens and comparison with conventional solvent extraction in the cytosol can lead to cell death (Popa et al., 2010). Antiox- methods. Comptes Rendus Chim 19, 692–698. idant scavengers of R. canina modulated and reduced the Boutennoun, H., Boussouf, L., Rawashdeh, A., Al-qaoud, K., oxidative stress of ROS to cells (HepG2 and SH-SY5Y). Abdelhafez, S., Kebieche, M., Madani, K., 2017. In vitro cytotoxic Three concentrations of ethanolic extract (7, 3.6 and 1.63 and antioxidant activities of phenolic components of Algerian lg/ml) containing t-BOOH similar to the control positive Achilleaodorata leaves. Arab. J. Chem. 10, 403–409. and without t-BOOH being lower to the negative control were Chaouche, T.M., Haddouchi, F., Ksouri, R., Atik-Bekkara, F., 2014. added for both cell types respectively. Results showed a reduc- Evaluation of antioxidant activity of hydromethanolic extracts of tion in the production of ROS in all concentrations of extract some medicinal species from South Algeria. J. Chinese Med. Assoc. used in this test compared to positive and negative controls for 77, 302–307. SH-SY5Y cells Fig. 7 (Jime´ nez et al., 2016). Moreover, the Cuyckens, F., Claeys, M., 2004. Mass spectrometry in the structural analysis of flavonoids. J. Mass Spectrom. 39, 1–15. concentrations of 7 and 3.6 lg/ml showed high reduction in F.Fernandes, Andrade, P.B., Ferreres, F., Gil-Izquierdo, A., Sousa- ROS in HepG2 compared to controls. Nevertheless, a concen- Pinto, I., Valenta˜ o, P., 2017. The chemical composition on tration of 1.63 lg/ml with tert-butyl resulted in lower reduc- fingerprint of Glandoradiffusa and its biological properties. Arab. tion of ROS compared to the positive control. It is J. Chem. 10, 583–595. noteworthy to mention that this oxidative effect of R. canina Friedrich, W., Eberhardt, A., 2000. Investigation of proanthocyanidins has never been mentioned before. The presence of sesquiter- by HPLC with electrospray ionization mass spectrometry. Eur. pene (bilobalide A) confers on the one hand neuroprotective Food Res. Technol., 56–64 effects and, on the other hand, the presence of such a tert- Granado-Serrano, A.B., Martı´ n, M.A., Bravo, L., Goya, L., Ramos, butyl group remains exceptional for such a natural compound. S., 2006. Quercetin induces apoptosis via caspase activation, This tert-butyl group was the major cause of the oxidative regulation of Bcl-2, and inhibition of PI-3-kinase/Akt and ERK pathways in a human hepatoma cell line (HepG2). J. Nutr. 136, effect of R. canina at this concentration. 2715–2721. There are an association of ROS and free radicals in the Jafri, L., Saleem, S., Ihsan-ul-Haq, Ullah, N., Mirza, B., 2017. In vitro pathogenesis of certain human sicknesses, including cancer, assessment of antioxidant potential and determination of poly- is ending progressively, in this study, the antioxidants of R. phenolic compounds of Hederanepalensis K. Koch.. Arab. J. canina fruits were modulated the ROS in order to reduce the Chem. 10, S3699–S3706. oxidative stress in HepG2 and SH-SY5Y cells and as a result Jime´ nez, S., Gasco´ n, S., Luquin, A., Laguna, M., Ancin-Azpilicueta, reduced the danger of risk in injury of cancer (Al-Gubory C., Rodrı´ guez-Yoldi, M.J., 2016. Rosa canina extracts have et al., 2010; Serrano et al., 2007; Trachootham et al., 2009). antiproliferative and antioxidant effects on caco-2 human colon cancer. PLoS One 11, 1–14. Kassim, M., Achoui, M., Mustafa, M.R., Mohd, M.A., Yusoff, K.M., 4. Conclusions 2010. Ellagic acid, phenolic acids, and flavonoids in Malaysian honey extracts demonstrate in vitro anti-inflammatory activity. In the present study, a number of analysis using HPLC-UV- Nutr. Res. 30, 650–659. MS method were carried out to reveal the composition of R. Khanam, U.K.S., Oba, S., Yanase, E., Murakami, Y., 2012. Phenolic canina. Afterwards, in situ essays on HepG2 and SH-SY5Y acids, flavonoids and total antioxidant capacity of selected leafy cells were carried out to further explore the antioxidative vegetables. J. Funct. Foods 4, 979–987. Lester, G.E., Lewers, K.S., Medina, M.B., Saftner, R.A., 2012. and cytotoxic effects of R. canina. Results indicated that the Comparative analysis of strawberry total phenolics via Fast Blue antioxidant and antiproliferative effects of R. canina fruits Composition and biological activity of the Algerian plant 1119

BB vs. Folin-Ciocalteu: Assay interference by ascorbic acid. J. Incarvillea emodi. Nat. Prod. Res.: Formerly Nat. Prod. Lett., 37– Food Compos. Anal. 27, 102–107. 41 Ma, J., Luo, X., Protiva, P., Yang, H., Ma, C., Basile, M.J., Weinstein, Ren, W., Qiao, Z., Wang, H., Zhu, L., Zhang, L., 2003. Flavonoids: I.B., Kennelly, E.J., 2003. Bioactive novel polyphenols from the Promising anticancer agents. Med. Res. Rev. 23, 519–534. fruit of Manilkara zapota (Sapodilla). J. Nat. Prod., 983–986 Ren, S.C., Qiao, Q.Q., Ding, X.L., 2013. Antioxidative activity of five Maria, R., Shirley, M., Xavier, C., Jaime, S., David, V., Rosa, S., flavones glycosides from corn silk (Stigma maydis). Czech J. Food Jodie, D., 2017. Preliminary screening, total phe- Sci. 31, 148–155. nolic content and antibacterial activity of thirteen native species Ridder, L., Van Der Hooft, J.J.J., Verhoeven, S., De Vos, R.C.H., Van from Guayas province Ecuador. J. King Saud Univ. – Sci. Schaik, R., Vervoort, J., 2012. Substructure-based annotation of Mcdunn, J.E., Li, Z., Adam, K., Neri, B.P., Wolfert, R.L., Milburn, high-resolution multistage MSn spectral trees. Rapid. Commun. M.V., Lotan, Y., Wheeler, T.M., 2013. Metabolomic signatures of Mass Spectrom. 26, 2461–2471. aggressive prostate cancer. Prostate, 1560. Shimada, K., Fujikawa, K., Yahara, K., Nakamura, T., 1992. Medina, M.B., 2011a. Simple and rapid method for the analysis of Antioxidative properties of xanthan on the autoxidation of phenolic compounds in beverages and grains. J. Agric. Food Chem. soybean oil in cyclodextrin emulsion. J. Agric. Food Chem. 40, 59, 1565–1571. 945–948. Medina, M.B., 2011b. Determination of the total phenolics in juices Serrano, J., Gon˜ i, I., Saura-Calixto, F., 2007. Food antioxidant and superfruits by a novel chemical method. J. Funct. Foods 3, 79– capacity determined by chemical methods may underestimate the 87. physiological antioxidant capacity. Food Res. Int. 40, 15–21. Montazeri, N., Baher, E., Mirzajani, F., Barami, Z., Yousefian, S., Talla, E., Tamfu, A.N., Gade, I.S., Yanda, L., Mbafor, J.T., Laurent, 2011. Phytochemical contents and biological activities of Rosa S., Elst, L.V., Popova, M., Bankova, V., 2016. New mono-ether of canina fruit from Iran. J. Med. Plants Res. 5, 4584–4589. glycerol and triterpenes with DPPH radical scavenging activity Mraihi, F., Hidalgo, M., de Pascual-Teresa, S., Trabelsi-Ayadi, M., from Cameroonian propolis. Nat. Prod. Res. 6419, 1–11. Che´ rif, J.K., 2015. Wild grown red and yellow hawthorn fruits Tchouya, G.R.F., Barhe, T.A., 2016. Comparative study of the anti- from Tunisia as source of antioxidants. Arab. J. Chem. 8, 570–578. oxidant activity of the total polyphenols extracted from Hibiscus https://doi.org/10.1016/j.arabjc.2014.11.045. Sabdariffa L ., Glycine max L. Merr., yellow tea and red wine Naczk, M., Shahidi, F., 2004. Extraction and analysis of phenolics in through reaction with DPPH free radicals. Arab. J. Chem., 1–8 food. J. Chromatogr. A 1054, 95–111. Tohma, H., Ko¨ ksal, E., Kılıc¸ ,O¨ ., Alan, Y., Yılmaz, M., Gu¨ lc¸ in, I.,_ Nwaehujor, C.O., Ezeja, M.I., Udeh, N.E., Okoye, D.N., Udegbunam, Bursal, E., Alwasel, S., 2016. RP-HPLC/MS/MS analysis of the R.I., 2014. Anti-inflammatory and anti-oxidant activities of Mal- phenolic compounds, antioxidant and antimicrobial activities of lotus oppositifolius (Geisel) methanol leaf extracts. Arab. J. Chem. Salvia L Species. Antioxidants 5, 38. 7, 805–810. Trachootham, D., Alexandre, J., Huang, P., 2009. Targeting cancer Pallaufa, K., Rivas-Gonzalo, J.C., del Castillo, M.D., Cano, M.P., de cells by ROS-mediated mechanisms: a radical therapeutic Pascual-Teresa, S., 2008. Characterization of the antioxidant approach? Nat. Rev. Drug Discov. 8, 579–591. composition of strawberry tree (Arbutus unedo L.) fruits. J. Food Wang, G., Li, T., Deng, F., Li, Y., Ye, W., 2013. Five new phenolic Composition Anal. 21, 273–281. glycosides from Hedyotis scandens. Bioorg. Med. Chem. Lett. 23, Plazonic´ , Ana, Bucar, F., Malesˆ ,Zˆ eljan, Mornar, A., Nigovic´ , B., 1379–1382. Kujundẑic´ , N., 2009. Identification and quantification of flavonoids Yang, B., Academy, C., Jiali, Y., Academy, C., Yupeng, Z., Garden, and phenolic acids in burr parsley (caucalisplatycarpos L.), using B., Acade, C., 2016. The plant resources, structure characteristics, high-performance liquid chromatography with diode array detec- biological activities and synthesis of pyranoflavonoids. Curr. Med. tion and electrospray ionization mass spectrometry. Molecules 14, Chem. 2466–2490. Yin, P., Yang, L., Li, K., Fan, H., Xue, Q., Li, X., Sun, L., 2019. Popa, C.-V., Dumitru, I., Ruta, L.L., Danet, A.F., Farcasanu, I.C., Bioactive components and antioxidant activities of cup crude 2010. Exogenous oxidative stress induces Ca2+ release in the yeast extract and its four partially purified fractions by HPD-100 Saccharomyces cerevisiae. FEBS J. 277, 4027–4038. macroporous resin chromatography. Arab. J. Chem. 12, 249–261. Rana, A., Rana, S., Majeed, R., Singh, H.P., 2014. Comparative studies for screening of bioactive constituents from various parts of